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Water gradients across bovine lenses
E:*TJ. Eye Res. (1987) 45, 191-195
L E T T E R TO THE E D I T O R S
Water
Gradients
Across
Bovine
Lenses
T h e age d e p e n d e n c e o f t h e n o n - f r e e z e a b l e w a t e r c o n t e n t s h o w e d d i f f e r e n t t r e n d s in h u m a n ( L a h m , Lee a n d B e t t e l h e i m , 1985) t h a n in r a t lenses (Castoro a n d B e t t e l h e i m . 1986). A l t h o u g h t h e t o t a l w a t e r c o n t e n t in b o t h species d e c r e a s e s w i t h age, e s p e c i a l l y in t h e n u c l e u s , t h e n o n - f r e e z e a b l e ( b o u n d ) w a t e r c o n t e n t increases in t h e r a t lens a n d d e c r e a s e s in t h e h u m a n lens w i t h age. This s h o w e d d i f f e r e n t i n v o l v e m e n t o f s y n e r e s i s in t h e a g i n g .of t h e t w o species. I n b o t h of t h e a b o v e s t u d i e s lenses w e r e o b t a i n e d f r o m i n d i v i d u a l s o f d i f f e r e n t - a g e s and the water contents of the cortex, i n t e r m e d i a t e layer and the nucleus, were a n a l y s e d as a f u n c t i o n o f c h r o n o l o g i c a l age. H o w e v e r , t h e r e exists also a n i n t e r n a l age i n d i c a t o r in t h e lens, since t h e lens g r o w s t h r o u g h o u t life. M o s t b u t n o t all o f t h e g r o w t h is a c c o u n t e d for b y an i n c r e a s e in t h e c o r t e x (R.afferty. 1985). T h u s a g r a d i e n t g o i n g fi'om t h e a n t e r i o r (or posterior) s u r f a c e t o t h e c e n t e r o f t h e n u c l e u s a l o n g t h e visual a x i s p r o v i d e s a n d ' i n t e r n a l age g r a d i e n t '. M e a s u r e m e n t o f s u c h an ' i n t e r n a l age g r a d i e n t ' w o u l d e l i m i n a t e t h e v a r i a t i o n d u e to t h e i n d i v i d u a l i t y a n d d i f f e r e n t h i s t o r y o f t h e s a m p l e s . H o w e v e r , one m u s t be careful in c o m p a r i n g t h e " i n t e r n a l a g e ' i n d e x w i t h t h e c h r o n o l o g i c a l age o f t h e lens. N e i t h e r o f t h e m is a t r u e age i n d e x . Tim ' i n t e r n a l a g e ' i n d e x w o u l d t r u l y reflect t h e a g i n g process if t h e r e w o u l d be rio c o m m u n i c a t i o n s b e t w e e n t h e tiber'cells, no fluxes o f w a t e r , m e t a b o l i t e s , etc. across t h e m e m b r a n e . U n d e r t h o s e c o n d i t i o n s t h e first c o r t i c a l fiber cell w o u l d be z e r o - y e a r old a n d t h e n u c l e u s w o u l d be t h e c h r o n o l o g i c a l a g e ; t h e d i f f e r e n t w a t e r g r a d i e n t s b e t w e e n t h e m w o u l d reflect t h e a g i n g process. B u t b e c a u s e o f water" fluxes a c r o s s t h e m e m b r a n e s n e i t h e r t h e d i f f e r e n t w a t e r m o l e c u l e s in the n u c l e u s h a v e t h e c h r o n o l o g i c a l age o f t h e a n i m a l , n o r t h e e q u i l i b r i u m c o n d i t i o n s b e t w e e n t h e d i f f e r e n t w a t e r m o l e c u l e s m a y be t h e s a m e as p r e v a i l e d a t b i r t h . T h e c h r o n o l o g i c a l age o f t h e lens s i m i l a r l y can be r e f e r r e d o n l y to t h e n u c l e u s . B o v i n e e y e s ( 2 - y e a r s - o l d ) w e r e o b t a i n e d f r o m s l a u g h t e r h o u s e s . T h e lenses w e r e w o r k e d u p w i t h i n 3 hr. T h e lenses w e r e placed in a r e f r i g e r a t e d m i c r o t o m e a t --10°C a n d s e c t i o n e d f r o m a n t e r i o r (or p o s t e r i o r ) s u r f a c e t o t h e nucleus. Six- to t w e n t y - f i v e m i l l i g r a m s a m p l e s w e r e i m m e d i a t e l y a n d h e r m e t i c a l l y e n c a p s u l a t e d in p r e - w e i g h e d c o a t e d a l u m i n i u m s a m p l e p a n s . T h e s e w e r e used t o m e a s u r e t h e f r e e z e a b l e (free) w a t e r c o n t e n t o f t h e lens b y d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC). F o r t h e d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC) a h e r m e t i c a l l y sealed e ~ p t y c o a t e d a l u m i n i u m p a n s e r v e d as a . r e f e r e n c e . T h e s a m p l e a n d r e f e r e n c e p a n was j ) l a c e d in a DSC ( D u P o n t 990, D u P o n t : W i l m i n g t o n , D E ) cell a n d cooled to --30°(3 b y an e x t e r n a l d r y ice b a t h . DSC c u r v e s w e r e o b t a i n e d b y h e a t i n g t h e s a m p l e a t a p r o g r a m m e d r a t e o f 3°(3 rain -~ t o 30°C. T h e i n s t r u m e n t was c a l i b r a t e d w i t h a s a p p h i r e disc a n d t h e DSC cell c a l i b r a t i o n c o n s t a n t was o b t a i n e d p e r i o d i c a l l y . T h e DSC c u r v e s r e c o r d e d t h e d i f f e r e n t i a l h e a t flow (q) as a f u n c t i o n o f t i m e . T h e q w a s r e c o r d e d s i m u l t a n e o u s l y w i t h t w o d i f f e r e n t sensitivities, for e x a m p l e , 0 - 5 m V c m -~, high s e n s i t i v i t y , a n d 10 m V c m -1, low s e n s i t i v i t y . T h e a r e a u n d e r t h e c u r v e g i v e s t h e n u m b e r o f J o f h e a t used to m e l t t h e m e a s u r e d m a s s o f w a t e r . Since it w a s o u r i n t e n t i o n t o t r a n s l a t e t h e a r e a o f a n e n d o t h e r m (in J p e r g s a m p l e ) t o a c e r t a i n a m o u n t o f f r e e z e a b l e w a t e r p e r g s a m p l e , we r a n a c a l i b r a t i o n c u r v e w i t h distilled w a t e r a n d 0014-4835/87/070191 + 0 5 $3.00/0
© 1987 Academic Press Inc. (London) Limited
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w i t h a q u e o u s NaCI s o l u t i o n s w i t h d i f f e r e n t c o n c e n t r a t i o n s h a v i n g d i f f e r e n t m e l t i n g (freezing) p o i n t s ( B e t t e l h e i m , C h r i s t i a n a n d Lee, 1983). A f t e r t h e DSC m e a s u r e m e n t s , t h e t o t a l w a t e r c o n t e n t o f t h e lens was m e a s u r e d . T h e p a n s w e r e p u n c t u r e d , t a k i n g c a r e n o t to d i s t u r b t h e s a m p l e s . T h e p a n s w e r e n e x t p l a c e d in a t h e r m o g r a v i m e t r i c a n a l y s e r ( D u P o n t 951) a n d t h e t o t a l w a t e r c o n t e n t o f t h e s a m p l e s o b t a i n e d f r o m t h e w e i g h t loss w h i c h o c c u r r e d d u r i n g h e a t i n g t h e palls to a n d m a i n t a i n i n g t h e m a t 105°C. T h e n o n - f r e e z e a b l e w a t e r c o n t e n t was o b t a i n e d as t h e d i f f e r e n c e b e t w e e n t h e t o t a l a n d f r e e z e a b l e w a t e r c o n t e n t , a n d it was e x p r e s s e d as a percentage of the total w a t e r content.
WATER
GRA1)IENTS
ACR()SS
BOVINE
1,ENSES
193
The dependence of the different water contents on the position along tim visual axis was obtained by regression analysis using the Minitab II program in a P r i m e computer (Prime: Boston, MA). The statistical significance of each regression analysis was established by the standard Student's t test. 'I'A m,~: 1
Re.qression. analysis of lhe different water contents as a function, of distance ( i n r a m ) front the surface (anterior or posterior) of bovine lenses along the visual axis la, t~s No.
Intercept a
Slope h
S.D.
r~
3 4 5 it
Total w a t e r % of lens weight 79"4 -- 10'9 2"37 75"2 - - 8"55 1 "37 77"6 -- 3"86 0"23 74" ! -- 6'09 I '23 79'5 -- 5" ! 0 0'49 73"9 --5" ! 1 0"65
0"810* 0"867" 0"963" 0"891 * 0'948" 0"925*
I 2 3 4 5 6
F'reezable w a t e r % of lens weight 64-8 -- 12.7 3.27 59.3 -- 10.9 !- 16 64.5 -- 5.26 0.35 61-8 -- 7.86 1"71 68"5 -- 6"23 0"47 60"8 -- 5.57 ~ 0"46
0.752¢ (}.936" 0.954* ()'876~ 0"966" 0.967"
1 2
1 2 3 4 5
6 1 2 3 4 5
6
Non-freezable w a t e r % of lens weight 14"6 1"78 (H16 15"9 2-38 1"08 13" I 1"41 0"36 12"3 ! "77 ! "85 ! 1 "0 1 "07 (t"26 13"2 0'46 0"45
0"407§ 0"447~ 0"579" 0"234 0"741 0" 177
Non-fi'eezable w a t e r % of total water ! 6"6 8"06 2"58 19"3 8"64 1"35 15"6 3"72 0"59 ! 5"8 5" 19 2'44 12"5 3"58 0"38 16"2 3"10 0"70
0"6ti I "}" 0"872* 11"784" 0"601 ~: 0"937" 0"796¢
Statistically significant at, the 99-9(*), 99(t), .q5($), 90(§) % confidence levels. T h e t o t a l w a t e r c o n t e n t g r a d i e n t o f a t y p i c a l b o v i n e l e n s is g i v e n in ]~ig. 1, t h e f r e e z e a b l e w a t e r c o n t e n t g r a d i e n t in F i g . 2 a n d t h e n o n - f r e e z a b l e w a t e r c o n t e n t in F i g . 3. T h e c o r r e s p o n d i n g r e g r e s s i o n c o e f f i c i e n t s , s t a n d a r d d e v i a t i o n s a n d c o r r e l a t i o n c o e f f i c i e n t s a r e p r e s e n t e d in T a b l e 1 f o r e a c h o f t h e b o v i n e l e n s e s i n v e s t i g a t e d . The total water content (as percentage of the lens weight) has a negative gradient (i.e. d e c r e a s e s g o i n g f i ' o m c o r t e x t o n u c l e u s ) a s h a s b e e n s h o w n a l s o b y P h i l i p s o n ( 1 9 6 9 ) in r a t l e n s e s . T h i s i m p l i e s t h a t d u r i n g m a t u r a t i o n o f l e n s f i b e r s , w a t e r is l o s t to accommodate a d e n s e r p a c k i n g o f l e n s p r o t e i n s . S i m i l a r l y t h e f r e e z e a b l e (fi-ee) w a t e r c o n t e n t o f t h e l e n s f r o m c o r t e x t o n u c l e u s a l s o h a s a n e g a t i v e g r a d i e n t . T h i s in t u r n would indicate that whatever water was lost from the lens fibers during 'aging' was fl'eezeable (free) water. The non-freezeable (bound) water percentage of bovine lens, on
194
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t i l e otJier Jliind, Jlii,~ ll ilo.~iliv~. • 7 i - l i d i o i t t . i.t+. ili(,l-l,il.~t,s l~flilig fl'oin i.Ol'lt,x l ~ liUl,Jl,llS. "rhi,~ l n l s i t i v e g r t l d i e n t is evi~n l n o r o lironl)un(.ed wJlt, ii t h e non-tYeozettlJJe w a t e r i~ ~,alctuhll:i;d |is il llt, l'(.(,nl o f t i m w a t t , r ( . o l i t o l i t (Tiitllt, i). ()lie l l l l i y h l i e r l l r e t t h e i l o s i l i v e ~i'tldit, lil (iJ" ilOll-[Fl'l,t+ziiJih, wiilel" i.(int(,llt, il,~i li i:Oli,¢ei'sion ii~ rl'eozeliblo (J'l'O(t) wittt#r t o noii-|'rt, i~zl,illlle ( h o u n d w a t e r ) d u e t o t i l e llill.J¢lil~ l i t l l r o t i , in liioh,l.uh,.~, l']simoiltlly i r t h e iillel¢ing i,~ |in l i g g i ' e t ~ l t i o n lll'ol,e~s i h i ' o u g h e o v i i h , n t l l o n d i n g o r h l n - d i l l o l e etu. h l t e r i l c ' t i o n s , w a t e r n i o l e c u l e s t h a t had sonic ti'iinsJatiliniiJ-rotationai J'roiJdonl tJl'eViOtiSl,V w i l l IlL, e n t r a l ) p O d , lose these ri.l,edolns t o lnoi'(J r i g i d t i e d .~yinnil, ll'il.lll o i ' ~ t i i i i z t i t i o n a n d I)l;(~onle l i o n - ~ ' r e e z e a l j i o wtttOl', Tlili.~ iloL o n l y tile iil~gtitive t o l i l i wiit(~l' ~i'itllit,iiL b u t iil.~(i t h e l)(mitivl; llOlifreezeallle w i i t e l ' g r i i d i l ; i i t Pi'oln c o r t e x t o nuoJeils (<'ontl'iinlto,~ t o t h e t o t a l r e f r a c t i v e h i d e x g r a d i e n t t h a t is llet;t~.sstiry t o t h e r e | ' r a e i i v e l l o w e r o f lll~, lens ( B e t i e l h e h n . l!),%5), .411 illt.l'ell,~,(; in t h e non-fi'eezealde w a t e r Oolltellt |'i'Olit (~()l'tex t o n u c l e u s , in essence, m a k e s t h e t o t a l r e f r a c t i v e index g r a d i e n t s m a l l e r t h a n it w o u h i be i | ' o n l y t h e ehailge in t o t a l w a t e r i~ontont, w o u h l lie t h e e o n t r i l n i t i n g t h e t o r . T h i s s t u d y rl:-emlillasizes tilt; nel.'l.,ssit.y ill" ( i i s l i n ( , t i o n liet.wet,n elll'onologi(mi agillg o f t h e lens |is a w h o l e l i n d t h e a g i n g due: t o lens g r o w t h ~ l e l i l o n s t r a t e ( i in t h e c o r t e x to ntwleus gradients. A(;K N()W I,E I)(;M ENT
T h i s w o r k w a s SUPllorted b y an N I H g r a n t No. EY 02571.
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l{ettelheinL F. A. (1985). l)tlysieal basis o f lens transtmrelmy. In The O c . h . . l.e..~. ,%'h'm'l.re. Fum'lion aml l'allmlofl!L (Ed..~!aisel, H.). Pp. 2(i5-3(1(1. 31. l),;kker l t w : New Y . r k . i{ettelheim, I,'. A. (197 I). E . r l . ; r i m e . t a l l~hysical (.'hemi,slr!t. P. 149. Saumlers : l~hiladelllhia. i{t,ttelheim, F. A.. Christian. S. and l,ee, I,. K. (19g3). l)itl'erential st'aiming ,.alorimetrie lll{,i_lSUl'(-llll'lltS ()ll hlilnillt Jl'llS. (..'ll#'r. l~lff: 1~1".'~.2. ~(),'{--N. ( T a s t o r o . . l . A . alld l{ettelheim, !". A. (i!)8G). l ) i s t r i h u t i o n , f t h ( ; total and llon-t'reezable water in rat lenses. Exp. Eye Re.~'. 43, 185-91. l,ailln. I)., Lee, I,. K . and l~etteiheim, F. A. (1985). Age dependenve of fi'eezabh; and nonf'reezalfle water c(intent of normal human lenses. I,ce.st. Ophlhalmol. l'is. Nci. 26. 1162-5. Monk. G . S . (1963). Liffht. Prim:|ides aml ExlJerimr'nt.~ (2rid edn). Pp. 456-7. Dover l'uldishing The: New York i~hilipson. B. (1969). D i s t r i b u t i o n ot* l)rotein w i t h i n the normal rat lens. lnce.~l. Ophthalmol. 8, 258-70. Rafferty, N . S . (1985). Lens Morphoh~gy. In The Ocular Len.~, ,S'tructure, Fanclio~, a~td Palholoffy. (Ed. Maisel, H.). Pp. 1-60. M. l)ekker Inc: New York. Appendix The l)rocess that increases tile non-freezeable water content as one proceeds from c o r t e x t o n u c l e u s c a n be c a l l e d a n i n v e r s e s y n e r e s i s . I t i n f l u e n c e s tile r e f l - a c t i v e i n d e x Please send reprint requests to l)r F. A. Bettelheim
WATER GRADIENTS ACROSS BOVINE LENSES
195
of the lens along the visual axis. A qualitative appreciation of this can be seen from i h e following considerations: (1) The refractive index of water decreases when one goes from freezeable (free) water to non-freezeable (bound) water. The refractive index of free water a t 37°C is 1"331 while t h a t of bound water, assuming ice-like structure, is 1"309 (ordinary) and 1"3104 (extraordinary) with sodium light (Monk, 1963). (2) As a first approximation, we may assume t h a t the molar refractivities in the lens are additive. If so, the following equation m a y apply (Bettelheim, 1971): nsoln--1
n~ol, + 2
=
n ~ - - l M l ~_v n ~ - - l M 1 na2 - 1 M 3 P~°~nXln~+2 Pl n ~ + 2 P2 ~'Xana2+2 Pa
X~ M 1 + X 2 M 2 + XaM~
(1)
where u is the refractive index and the subscripts 1,2,3 and soln refer to the fi'ee water, bound water, lens proteins and to the lens, respectively. M is the molecular weight, X is the mole fi'action and p is the density. The density of the lens can be al)l)roximated by : ! = w__2+ 1 w_~+ w..~ (2) P~o~n
Pl
P.z
P3
where w is the weight fraction. The right hand side of equation (1) increases as the refi'active index of the lens increases. The first two terms of the n o m i n a t o r in e q u a t i o n (1) shows the c o n t r i b u t i o n of inverse syneresis to the change of the refractive index along the visual axis. In general, the inverse syneretic process lessens the increase in refractive index m o v i n g from cortex to nucleus, t h a t would occur if one considers only changes in the protein (,oncentrations along the visual axis. The m a g n i t u d e of such influence upon the refractive index g r a d i e n t due to inverse syneresis d e p e n d s on t h e relative contributions of the three terms of the n o m i n a t o r in e q u a t i o n (1). A sample calculation using the p a r a m e t e r s of lens No.3 in Table I and the following densities: 0'99336, 0"99986 and 1.30 for free water, bound water and lens protein, respectively, was performed. The other p a r a m e t e r s used were 1.331, 1.309 and 1"50 for the refractive indices of free water, bound water and lens proteins, respectively. The average molecular weiglit of the lens l)roteins was a p p r o x i m a t e d by using the value of 200000. The calculation yielded the following results: (1) ~¥ith inverse syneresis [equation (I)] tim refi'active index increases by 0"005 units tbr every m m distance on t h e cortex to nucleus axis. (2) W i t h o u t inverse syneresis (when the first two terms in the brackets of the n o m i n a t o r are combined and one uses only n I and Pl) the same calculation would yield an increase of 0"00764 units in the refi.active index per m m distance. This calculation should be taken only as an indication of the direction of the change contributed by the inverse syneresis to the refractive index gra(iient. The basic assumption, i.e. the a d d i t i v i t y of molar refractivities m a y n o t apply to the lens and the density, the refractive index and the molecular weight of the lens proteins used in this calculation m a y be in error. These, however, do not change the general conclusions t h a t inverse syneresis lessens the refractive index gradient produced by a change in protein concentrations, alone. In the above example, inverse syneresis exerted a 3 3 % change in the refi'active index gradient.
L E T T E R TO THE E D I T O R S
Water
Gradients
Across
Bovine
Lenses
T h e age d e p e n d e n c e o f t h e n o n - f r e e z e a b l e w a t e r c o n t e n t s h o w e d d i f f e r e n t t r e n d s in h u m a n ( L a h m , Lee a n d B e t t e l h e i m , 1985) t h a n in r a t lenses (Castoro a n d B e t t e l h e i m . 1986). A l t h o u g h t h e t o t a l w a t e r c o n t e n t in b o t h species d e c r e a s e s w i t h age, e s p e c i a l l y in t h e n u c l e u s , t h e n o n - f r e e z e a b l e ( b o u n d ) w a t e r c o n t e n t increases in t h e r a t lens a n d d e c r e a s e s in t h e h u m a n lens w i t h age. This s h o w e d d i f f e r e n t i n v o l v e m e n t o f s y n e r e s i s in t h e a g i n g .of t h e t w o species. I n b o t h of t h e a b o v e s t u d i e s lenses w e r e o b t a i n e d f r o m i n d i v i d u a l s o f d i f f e r e n t - a g e s and the water contents of the cortex, i n t e r m e d i a t e layer and the nucleus, were a n a l y s e d as a f u n c t i o n o f c h r o n o l o g i c a l age. H o w e v e r , t h e r e exists also a n i n t e r n a l age i n d i c a t o r in t h e lens, since t h e lens g r o w s t h r o u g h o u t life. M o s t b u t n o t all o f t h e g r o w t h is a c c o u n t e d for b y an i n c r e a s e in t h e c o r t e x (R.afferty. 1985). T h u s a g r a d i e n t g o i n g fi'om t h e a n t e r i o r (or posterior) s u r f a c e t o t h e c e n t e r o f t h e n u c l e u s a l o n g t h e visual a x i s p r o v i d e s a n d ' i n t e r n a l age g r a d i e n t '. M e a s u r e m e n t o f s u c h an ' i n t e r n a l age g r a d i e n t ' w o u l d e l i m i n a t e t h e v a r i a t i o n d u e to t h e i n d i v i d u a l i t y a n d d i f f e r e n t h i s t o r y o f t h e s a m p l e s . H o w e v e r , one m u s t be careful in c o m p a r i n g t h e " i n t e r n a l a g e ' i n d e x w i t h t h e c h r o n o l o g i c a l age o f t h e lens. N e i t h e r o f t h e m is a t r u e age i n d e x . Tim ' i n t e r n a l a g e ' i n d e x w o u l d t r u l y reflect t h e a g i n g process if t h e r e w o u l d be rio c o m m u n i c a t i o n s b e t w e e n t h e tiber'cells, no fluxes o f w a t e r , m e t a b o l i t e s , etc. across t h e m e m b r a n e . U n d e r t h o s e c o n d i t i o n s t h e first c o r t i c a l fiber cell w o u l d be z e r o - y e a r old a n d t h e n u c l e u s w o u l d be t h e c h r o n o l o g i c a l a g e ; t h e d i f f e r e n t w a t e r g r a d i e n t s b e t w e e n t h e m w o u l d reflect t h e a g i n g process. B u t b e c a u s e o f water" fluxes a c r o s s t h e m e m b r a n e s n e i t h e r t h e d i f f e r e n t w a t e r m o l e c u l e s in the n u c l e u s h a v e t h e c h r o n o l o g i c a l age o f t h e a n i m a l , n o r t h e e q u i l i b r i u m c o n d i t i o n s b e t w e e n t h e d i f f e r e n t w a t e r m o l e c u l e s m a y be t h e s a m e as p r e v a i l e d a t b i r t h . T h e c h r o n o l o g i c a l age o f t h e lens s i m i l a r l y can be r e f e r r e d o n l y to t h e n u c l e u s . B o v i n e e y e s ( 2 - y e a r s - o l d ) w e r e o b t a i n e d f r o m s l a u g h t e r h o u s e s . T h e lenses w e r e w o r k e d u p w i t h i n 3 hr. T h e lenses w e r e placed in a r e f r i g e r a t e d m i c r o t o m e a t --10°C a n d s e c t i o n e d f r o m a n t e r i o r (or p o s t e r i o r ) s u r f a c e t o t h e nucleus. Six- to t w e n t y - f i v e m i l l i g r a m s a m p l e s w e r e i m m e d i a t e l y a n d h e r m e t i c a l l y e n c a p s u l a t e d in p r e - w e i g h e d c o a t e d a l u m i n i u m s a m p l e p a n s . T h e s e w e r e used t o m e a s u r e t h e f r e e z e a b l e (free) w a t e r c o n t e n t o f t h e lens b y d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC). F o r t h e d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC) a h e r m e t i c a l l y sealed e ~ p t y c o a t e d a l u m i n i u m p a n s e r v e d as a . r e f e r e n c e . T h e s a m p l e a n d r e f e r e n c e p a n was j ) l a c e d in a DSC ( D u P o n t 990, D u P o n t : W i l m i n g t o n , D E ) cell a n d cooled to --30°(3 b y an e x t e r n a l d r y ice b a t h . DSC c u r v e s w e r e o b t a i n e d b y h e a t i n g t h e s a m p l e a t a p r o g r a m m e d r a t e o f 3°(3 rain -~ t o 30°C. T h e i n s t r u m e n t was c a l i b r a t e d w i t h a s a p p h i r e disc a n d t h e DSC cell c a l i b r a t i o n c o n s t a n t was o b t a i n e d p e r i o d i c a l l y . T h e DSC c u r v e s r e c o r d e d t h e d i f f e r e n t i a l h e a t flow (q) as a f u n c t i o n o f t i m e . T h e q w a s r e c o r d e d s i m u l t a n e o u s l y w i t h t w o d i f f e r e n t sensitivities, for e x a m p l e , 0 - 5 m V c m -~, high s e n s i t i v i t y , a n d 10 m V c m -1, low s e n s i t i v i t y . T h e a r e a u n d e r t h e c u r v e g i v e s t h e n u m b e r o f J o f h e a t used to m e l t t h e m e a s u r e d m a s s o f w a t e r . Since it w a s o u r i n t e n t i o n t o t r a n s l a t e t h e a r e a o f a n e n d o t h e r m (in J p e r g s a m p l e ) t o a c e r t a i n a m o u n t o f f r e e z e a b l e w a t e r p e r g s a m p l e , we r a n a c a l i b r a t i o n c u r v e w i t h distilled w a t e r a n d 0014-4835/87/070191 + 0 5 $3.00/0
© 1987 Academic Press Inc. (London) Limited
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w i t h a q u e o u s NaCI s o l u t i o n s w i t h d i f f e r e n t c o n c e n t r a t i o n s h a v i n g d i f f e r e n t m e l t i n g (freezing) p o i n t s ( B e t t e l h e i m , C h r i s t i a n a n d Lee, 1983). A f t e r t h e DSC m e a s u r e m e n t s , t h e t o t a l w a t e r c o n t e n t o f t h e lens was m e a s u r e d . T h e p a n s w e r e p u n c t u r e d , t a k i n g c a r e n o t to d i s t u r b t h e s a m p l e s . T h e p a n s w e r e n e x t p l a c e d in a t h e r m o g r a v i m e t r i c a n a l y s e r ( D u P o n t 951) a n d t h e t o t a l w a t e r c o n t e n t o f t h e s a m p l e s o b t a i n e d f r o m t h e w e i g h t loss w h i c h o c c u r r e d d u r i n g h e a t i n g t h e palls to a n d m a i n t a i n i n g t h e m a t 105°C. T h e n o n - f r e e z e a b l e w a t e r c o n t e n t was o b t a i n e d as t h e d i f f e r e n c e b e t w e e n t h e t o t a l a n d f r e e z e a b l e w a t e r c o n t e n t , a n d it was e x p r e s s e d as a percentage of the total w a t e r content.
WATER
GRA1)IENTS
ACR()SS
BOVINE
1,ENSES
193
The dependence of the different water contents on the position along tim visual axis was obtained by regression analysis using the Minitab II program in a P r i m e computer (Prime: Boston, MA). The statistical significance of each regression analysis was established by the standard Student's t test. 'I'A m,~: 1
Re.qression. analysis of lhe different water contents as a function, of distance ( i n r a m ) front the surface (anterior or posterior) of bovine lenses along the visual axis la, t~s No.
Intercept a
Slope h
S.D.
r~
3 4 5 it
Total w a t e r % of lens weight 79"4 -- 10'9 2"37 75"2 - - 8"55 1 "37 77"6 -- 3"86 0"23 74" ! -- 6'09 I '23 79'5 -- 5" ! 0 0'49 73"9 --5" ! 1 0"65
0"810* 0"867" 0"963" 0"891 * 0'948" 0"925*
I 2 3 4 5 6
F'reezable w a t e r % of lens weight 64-8 -- 12.7 3.27 59.3 -- 10.9 !- 16 64.5 -- 5.26 0.35 61-8 -- 7.86 1"71 68"5 -- 6"23 0"47 60"8 -- 5.57 ~ 0"46
0.752¢ (}.936" 0.954* ()'876~ 0"966" 0.967"
1 2
1 2 3 4 5
6 1 2 3 4 5
6
Non-freezable w a t e r % of lens weight 14"6 1"78 (H16 15"9 2-38 1"08 13" I 1"41 0"36 12"3 ! "77 ! "85 ! 1 "0 1 "07 (t"26 13"2 0'46 0"45
0"407§ 0"447~ 0"579" 0"234 0"741 0" 177
Non-fi'eezable w a t e r % of total water ! 6"6 8"06 2"58 19"3 8"64 1"35 15"6 3"72 0"59 ! 5"8 5" 19 2'44 12"5 3"58 0"38 16"2 3"10 0"70
0"6ti I "}" 0"872* 11"784" 0"601 ~: 0"937" 0"796¢
Statistically significant at, the 99-9(*), 99(t), .q5($), 90(§) % confidence levels. T h e t o t a l w a t e r c o n t e n t g r a d i e n t o f a t y p i c a l b o v i n e l e n s is g i v e n in ]~ig. 1, t h e f r e e z e a b l e w a t e r c o n t e n t g r a d i e n t in F i g . 2 a n d t h e n o n - f r e e z a b l e w a t e r c o n t e n t in F i g . 3. T h e c o r r e s p o n d i n g r e g r e s s i o n c o e f f i c i e n t s , s t a n d a r d d e v i a t i o n s a n d c o r r e l a t i o n c o e f f i c i e n t s a r e p r e s e n t e d in T a b l e 1 f o r e a c h o f t h e b o v i n e l e n s e s i n v e s t i g a t e d . The total water content (as percentage of the lens weight) has a negative gradient (i.e. d e c r e a s e s g o i n g f i ' o m c o r t e x t o n u c l e u s ) a s h a s b e e n s h o w n a l s o b y P h i l i p s o n ( 1 9 6 9 ) in r a t l e n s e s . T h i s i m p l i e s t h a t d u r i n g m a t u r a t i o n o f l e n s f i b e r s , w a t e r is l o s t to accommodate a d e n s e r p a c k i n g o f l e n s p r o t e i n s . S i m i l a r l y t h e f r e e z e a b l e (fi-ee) w a t e r c o n t e n t o f t h e l e n s f r o m c o r t e x t o n u c l e u s a l s o h a s a n e g a t i v e g r a d i e n t . T h i s in t u r n would indicate that whatever water was lost from the lens fibers during 'aging' was fl'eezeable (free) water. The non-freezeable (bound) water percentage of bovine lens, on
194
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t i l e otJier Jliind, Jlii,~ ll ilo.~iliv~. • 7 i - l i d i o i t t . i.t+. ili(,l-l,il.~t,s l~flilig fl'oin i.Ol'lt,x l ~ liUl,Jl,llS. "rhi,~ l n l s i t i v e g r t l d i e n t is evi~n l n o r o lironl)un(.ed wJlt, ii t h e non-tYeozettlJJe w a t e r i~ ~,alctuhll:i;d |is il llt, l'(.(,nl o f t i m w a t t , r ( . o l i t o l i t (Tiitllt, i). ()lie l l l l i y h l i e r l l r e t t h e i l o s i l i v e ~i'tldit, lil (iJ" ilOll-[Fl'l,t+ziiJih, wiilel" i.(int(,llt, il,~i li i:Oli,¢ei'sion ii~ rl'eozeliblo (J'l'O(t) wittt#r t o noii-|'rt, i~zl,illlle ( h o u n d w a t e r ) d u e t o t i l e llill.J¢lil~ l i t l l r o t i , in liioh,l.uh,.~, l']simoiltlly i r t h e iillel¢ing i,~ |in l i g g i ' e t ~ l t i o n lll'ol,e~s i h i ' o u g h e o v i i h , n t l l o n d i n g o r h l n - d i l l o l e etu. h l t e r i l c ' t i o n s , w a t e r n i o l e c u l e s t h a t had sonic ti'iinsJatiliniiJ-rotationai J'roiJdonl tJl'eViOtiSl,V w i l l IlL, e n t r a l ) p O d , lose these ri.l,edolns t o lnoi'(J r i g i d t i e d .~yinnil, ll'il.lll o i ' ~ t i i i i z t i t i o n a n d I)l;(~onle l i o n - ~ ' r e e z e a l j i o wtttOl', Tlili.~ iloL o n l y tile iil~gtitive t o l i l i wiit(~l' ~i'itllit,iiL b u t iil.~(i t h e l)(mitivl; llOlifreezeallle w i i t e l ' g r i i d i l ; i i t Pi'oln c o r t e x t o nuoJeils (<'ontl'iinlto,~ t o t h e t o t a l r e f r a c t i v e h i d e x g r a d i e n t t h a t is llet;t~.sstiry t o t h e r e | ' r a e i i v e l l o w e r o f lll~, lens ( B e t i e l h e h n . l!),%5), .411 illt.l'ell,~,(; in t h e non-fi'eezealde w a t e r Oolltellt |'i'Olit (~()l'tex t o n u c l e u s , in essence, m a k e s t h e t o t a l r e f r a c t i v e index g r a d i e n t s m a l l e r t h a n it w o u h i be i | ' o n l y t h e ehailge in t o t a l w a t e r i~ontont, w o u h l lie t h e e o n t r i l n i t i n g t h e t o r . T h i s s t u d y rl:-emlillasizes tilt; nel.'l.,ssit.y ill" ( i i s l i n ( , t i o n liet.wet,n elll'onologi(mi agillg o f t h e lens |is a w h o l e l i n d t h e a g i n g due: t o lens g r o w t h ~ l e l i l o n s t r a t e ( i in t h e c o r t e x to ntwleus gradients. A(;K N()W I,E I)(;M ENT
T h i s w o r k w a s SUPllorted b y an N I H g r a n t No. EY 02571.
l)ep.rlmenl of ('hvmi.uh'!I,
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l{ettelheinL F. A. (1985). l)tlysieal basis o f lens transtmrelmy. In The O c . h . . l.e..~. ,%'h'm'l.re. Fum'lion aml l'allmlofl!L (Ed..~!aisel, H.). Pp. 2(i5-3(1(1. 31. l),;kker l t w : New Y . r k . i{ettelheim, I,'. A. (197 I). E . r l . ; r i m e . t a l l~hysical (.'hemi,slr!t. P. 149. Saumlers : l~hiladelllhia. i{t,ttelheim, F. A.. Christian. S. and l,ee, I,. K. (19g3). l)itl'erential st'aiming ,.alorimetrie lll{,i_lSUl'(-llll'lltS ()ll hlilnillt Jl'llS. (..'ll#'r. l~lff: 1~1".'~.2. ~(),'{--N. ( T a s t o r o . . l . A . alld l{ettelheim, !". A. (i!)8G). l ) i s t r i h u t i o n , f t h ( ; total and llon-t'reezable water in rat lenses. Exp. Eye Re.~'. 43, 185-91. l,ailln. I)., Lee, I,. K . and l~etteiheim, F. A. (1985). Age dependenve of fi'eezabh; and nonf'reezalfle water c(intent of normal human lenses. I,ce.st. Ophlhalmol. l'is. Nci. 26. 1162-5. Monk. G . S . (1963). Liffht. Prim:|ides aml ExlJerimr'nt.~ (2rid edn). Pp. 456-7. Dover l'uldishing The: New York i~hilipson. B. (1969). D i s t r i b u t i o n ot* l)rotein w i t h i n the normal rat lens. lnce.~l. Ophthalmol. 8, 258-70. Rafferty, N . S . (1985). Lens Morphoh~gy. In The Ocular Len.~, ,S'tructure, Fanclio~, a~td Palholoffy. (Ed. Maisel, H.). Pp. 1-60. M. l)ekker Inc: New York. Appendix The l)rocess that increases tile non-freezeable water content as one proceeds from c o r t e x t o n u c l e u s c a n be c a l l e d a n i n v e r s e s y n e r e s i s . I t i n f l u e n c e s tile r e f l - a c t i v e i n d e x Please send reprint requests to l)r F. A. Bettelheim
WATER GRADIENTS ACROSS BOVINE LENSES
195
of the lens along the visual axis. A qualitative appreciation of this can be seen from i h e following considerations: (1) The refractive index of water decreases when one goes from freezeable (free) water to non-freezeable (bound) water. The refractive index of free water a t 37°C is 1"331 while t h a t of bound water, assuming ice-like structure, is 1"309 (ordinary) and 1"3104 (extraordinary) with sodium light (Monk, 1963). (2) As a first approximation, we may assume t h a t the molar refractivities in the lens are additive. If so, the following equation m a y apply (Bettelheim, 1971): nsoln--1
n~ol, + 2
=
n ~ - - l M l ~_v n ~ - - l M 1 na2 - 1 M 3 P~°~nXln~+2 Pl n ~ + 2 P2 ~'Xana2+2 Pa
X~ M 1 + X 2 M 2 + XaM~
(1)
where u is the refractive index and the subscripts 1,2,3 and soln refer to the fi'ee water, bound water, lens proteins and to the lens, respectively. M is the molecular weight, X is the mole fi'action and p is the density. The density of the lens can be al)l)roximated by : ! = w__2+ 1 w_~+ w..~ (2) P~o~n
Pl
P.z
P3
where w is the weight fraction. The right hand side of equation (1) increases as the refi'active index of the lens increases. The first two terms of the n o m i n a t o r in e q u a t i o n (1) shows the c o n t r i b u t i o n of inverse syneresis to the change of the refractive index along the visual axis. In general, the inverse syneretic process lessens the increase in refractive index m o v i n g from cortex to nucleus, t h a t would occur if one considers only changes in the protein (,oncentrations along the visual axis. The m a g n i t u d e of such influence upon the refractive index g r a d i e n t due to inverse syneresis d e p e n d s on t h e relative contributions of the three terms of the n o m i n a t o r in e q u a t i o n (1). A sample calculation using the p a r a m e t e r s of lens No.3 in Table I and the following densities: 0'99336, 0"99986 and 1.30 for free water, bound water and lens protein, respectively, was performed. The other p a r a m e t e r s used were 1.331, 1.309 and 1"50 for the refractive indices of free water, bound water and lens proteins, respectively. The average molecular weiglit of the lens l)roteins was a p p r o x i m a t e d by using the value of 200000. The calculation yielded the following results: (1) ~¥ith inverse syneresis [equation (I)] tim refi'active index increases by 0"005 units tbr every m m distance on t h e cortex to nucleus axis. (2) W i t h o u t inverse syneresis (when the first two terms in the brackets of the n o m i n a t o r are combined and one uses only n I and Pl) the same calculation would yield an increase of 0"00764 units in the refi.active index per m m distance. This calculation should be taken only as an indication of the direction of the change contributed by the inverse syneresis to the refractive index gra(iient. The basic assumption, i.e. the a d d i t i v i t y of molar refractivities m a y n o t apply to the lens and the density, the refractive index and the molecular weight of the lens proteins used in this calculation m a y be in error. These, however, do not change the general conclusions t h a t inverse syneresis lessens the refractive index gradient produced by a change in protein concentrations, alone. In the above example, inverse syneresis exerted a 3 3 % change in the refi'active index gradient.